The Bcl-2 family proteins regulate the mitochondrial pathway of programmed cell death (apoptosis). The anti- apoptotic members, including Bcl-2, are survival proteins that are elevated in many cancers including prostate, breast and lung cancers as well as leukemia's. Bcl-2 blocks pro-apoptotic family members by binding their BH3 domains leading cancer drug researchers to search for inhibitors of this interaction. Recently, we demonstrated that Bcl-2 could be converted from a protector to a killer of cancer cells by Nur77, a nuclear orphan receptor, which can be induced to migrate from the nucleus to cytoplasm to convert Bcl-2 to a killer. Nur77 induces a Bcl-2 conformational change, exposing Bcl-2's latently pro-apoptotic BH3 domain, which induces the apoptosis of cancer cells. In addition, we identified a 9 amino acid Nur77-based peptide, NuBCP-9, that mimics Nur77 converter activity by not only inactivating Bcl-2's anti-apoptotic function but also converting it to a killer. This may open the way to a novel class of cancer drugs, small molecule Bcl-2 converters that may prove effective against late stage cancers, often characterized by elevated levels of Bcl-2. We aim to develop a primary fluorescent polarization competition assay and a secondary (alternative) time-resolved fluorescence resonance energy transfer (TR-FRET) competition assay to identify small molecule inhibitors of fluorescently labeled- NuBCP-9 binding to Bcl-2 and evaluate them for high-throughput screening using the 1280 member LOPAC library. Further, we aim to develop and validate additional secondary biophysical and cell-based assays for identifying those "hits" that induce the Bcl-2 conformational change characteristic of its'pro-apoptotic state and induce killing of breast cancer, prostate cancer, lung cancer and leukemia cell lines. PUBLIC HEALTH RELEVANCE: Bcl-2 is a survival protein that is over expressed in many late stage cancers including breast, prostate, lung and leukemia's. This project will establish and evaluate assays for the high-throughput screening of small molecules that convert Bcl-2 from a protector to a killer for treating late-stage cancers.